The p53 tumor suppressor plays critical roles in both cancer-prevention and aging. Activation of p53 response is important for its tumor suppressor function;conversely, shutting off active p53 is critical to prevent premature aging. This dual role of p53 in cancer and aging is perhaps best demonstrated by the activity of a naturally-occurring isoform of p53 called Np53 (a.k.a. 40p53). When over-expressed in mice (together with wild-type p53), Np53 causes hyper-activation of p53 target genes;although this results in enhanced tumor suppression, the mice also age rapidly and die prematurely. The hyper-active p53 response caused by Np53 has been linked to transcription activation. Because p53 binds DNA and activates transcription as a tetramer, the formation of p53/ Np53 hetero-tetramers is believed to cause the enhanced gene expression and accelerated aging. However, the molecular mechanism by which this occurs is largely unexplored. In this proposal, we outline a series of experiments that will begin to define the molecular mechanisms by which Np53 functions within the context of the p53 tetramer. Central to this work is a detailed biochemical analysis of mixed p53/ Np53 tetramers in the context of a native p53 target gene using a reconstituted human transcription system. In addition, we will identify which p53 target genes are differentially impacted by p53/ Np53 in human cells by performing mRNA analysis of 28 known p53 target genes. We will focus specifically on aging-related p53 targets in this screen to determine which might be altered most significantly by the Np53 isoform. Taken together, these biochemical and cell-based studies will begin to define the molecular mechanisms that enable the Np53 isoform to accelerate mammalian aging. PUBLIC HEALTH RELEVANCE: Our research analyzes the basic mechanisms of gene expression-what turns a gene "on" or "off" in a cell. Understanding this process is vital because proper regulation of gene expression is essential for virtually every major physiological process;furthermore, breakdown in this regulation is a hallmark of human disease, most notably cancer. In this proposal we will examine the function of an improperly activated protein called p53. Understanding how p53 functions, particularly in its abnormal, "hyper-active" state, lies at the heart of the cancer and aging problem because the form of p53 we are studying shows enhanced ability to prevent cancer yet concomitantly shortens lifespan. The precise mechanisms by which this occurs are not at all understood and will be explored in this study. We anticipate that the information accumulated by our efforts will identify new strategies for controlling the transcriptional activity of p53.